This invention relates to certain dinucleotides which increase the hydration of retained mucus secretions, stimulate the production of mucins and increase ciliary beat frequency to increase clearance of retained secretions.
Chronic obstructive pulmonary disease (COPD) affects 15 million patients in the U.S. and is the sixth leading cause of death. It is characterized by the retention of mucus secretions in the lungs. Many patients diagnosed with COPD have a disorder called chronic bronchitis (CB), and 600,000 patients are hospitalized each year due to an acute exacerbation of CB. Cystic fibrosis and Primary Ciliary Dyskinesia (PCD) are other examples of lung disorders which assume a clinical profile similar to COPD. Ciliary dyskinesia, whether primary or secondary, results in retained secretions that can only be cleared by coughing.
Another disease state characterized by the accumulation of retained mucous secretions is sinusitis. Sinusitis is an inflammation of the paranasal sinuses typically associated with an upper respiratory infection. It is this country""s most common health-care complaint, affecting an estimated 31 million people. (A. Moss and V. Parsons, National Center for Health Statistics, 1986: 66-7, DHHS Publication No. (PHS)86-1588 (1985)).
Otitis media (OM) is a viral or bacterial infection of the middle ear which primarily afflicts children under the age of three. It is usually precipitated by an upper respiratory infection which spreads into the middle ear via the nasopharynx and eustachian tube. Approximately 25-50 million office visits are made each year for diagnosis and treatment of OM. By age three, about 75% of children will have had at least one episode of acute OM (J. Klein, Clin. Infect. Dis. 19, 823-33 (1994)). Following appropriate treatment with antibiotics, accumulated fluid in the middle ear remains, causing hearing impairment and potential language and cognitive development delays. Enhanced ability to clear secretions in the middle ear would reduce or eliminate significant sequelae of otitis media.
An additional disorder resulting from retained secretions is pneumonia. Patients who are immobilized for a variety of reasons are at high risk for developing pneumonia. Despite extra vigilance and numerous interventions, pneumonia develops in over 400,000 patients per year, with significant morbidity and mortality.
There are also situations where it is therapeutically desirable to increase drainage of the lacrimal system. When the lacrimal drainage system is not functioning properly the result can be excessive tearing (epiphora), mucopurulent discharge, and recurrent dacryocystitis. Current treatments for nasolacrimal duct obstruction are mostly invasive surgical procedures, and researchers have sought to discover noninvasive pharmaceutical treatments.
Tear secretion can be stimulated from lacrimal accessory tissues via P2Y2 and/or P2Y4 purinergic receptor-mediated mechanisms similar to those which hydrate airway epithelia. Dry eye disease is the general term for indications produced by abnormalities of the precomeal tear film characterized by a decrease in tear production or an increase in tear film evaporatioin, together with the ocular surface disease that results. Currently, the pharmaceutical treatment of dry eye disease is mostly limited to administration of artificial tears (saline solution) to temporarily rehydrate the eyes. However, relief is short lived and frequent dosing is necessary.
Normally, mucous secretions are removed via the mucociliary clearance (MCC) system. MCC relies on the integrated action of three components: 1) mucus secretion by goblet cells and submucosal glands; 2) the movement of cilia on epithelial cells which propels the mucus across the luminal surface; and 3) ion transport into and out of luminal epithelial cells which concomitantly controls the flow of water into the mucus.
It is now known that nucleoside phosphates such as uridine 5xe2x80x2-triphosphate (UTP) modulate all of the components of the MCC system. First, UTP has been shown to increase both the rate and total amount of mucin secretion by goblet cells in vitro (M. Lethem, et al., Am J Respir. Cell Mol. Biol 9, 315-22 (1993)). Second, UTP has been shown to increase cilia beat frequency in human airway epithelial cells in vitro (D. Drutz, et al., Drug Dev. Res. 37(3), 185 (1996)). And third, UTP has been shown to increase Clxe2x88x92 secretion, and hence, water secretion from airway epithelial cells in vitro (S. Mason, et al., Br. J Pharmacol. 103, 1649-56 (1991)). In addition, it is thought that the release of surfactant from Type II alveolar cells in response to UTP (Gobran, Am. J. Physiol 267, L625-L633 (1994)) contributes to optimal functioning of the lungs and may assist in maximizing MCC (M. Knowles, et al., N. Engl. J. Med. 325, 533-38 (1991)). UTP has been shown to increase intracellular Ca++ due to stimulation of phospholipase C by the P2Y2 receptor (H. Brown, et al., Mol. Pharmacol 40, 648-55 (1991)).
UTP""s modulation of all components of the mucociliary escalator system results in a 2.5-fold improvement in lung mucociliary clearance in normal volunteers without any significant side-effects (K. Olivier, et al., Am J. Respir. Crit. Care Med. 154, 217-23 (1996)). In addition, UTP significantly enhanced cough clearance (clearance of retained secretions by coughing) in patients with PCD (P. Noone, et al., Am. J. Respir. Crit. Care Med. 153, A530 (1996)).
Because of UTP""s demonstrated ability to increase the clearance of retained mucous secretions, applicants were motivated to investigate whether other nucleoside phosphates could be equally, if not more, therapeutically effective. The present invention is based upon this investigation.
Previously described dinucleotides are listed in Table I, along with their corresponding literature references.
(1) M. A. G. Sillero et al., Eur. J. Biochem., 76, 331 (1997)
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(8) P.C. Zamecnik et al., Proc. Natl. Acad. Sci., 89, 2370 (1992)
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(17) G. Klein et al., Biochemistry, 27, 1897 (1988)
(18) E. Castro et al., Br. J. Pharmacol., 100, 360 (1990)
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(23) A. Guranowski et al., Biochemistry, 27, 2959 (1988)
(24) F. Grummt et al., Plant Mol. Bio., 2, 41 (1983)
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(27) E. Rapaport et al., Proc. Natl. Acad. Sci., 78, 838 (1981)
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(29) E. Schulze-Lohoffet al., Hypertension, 26, 899 (1995)
(30) B. K. Kim et al., Proc. Natl. Acad. Sci., 89, 11056 (1992)
(31) P. C. Zamecnik et al., Proc. Natl. Acad. Sci., 89, 2370 (1992)
(32) H. Morii et al., Eur. J. Biochem., 205, 979 (1992)
(33) E. Castro et al., Pflugers Arch., 426, 524 (1994)
(34) H. Schluter et al., Nature, 367, 186 (1994)
(35) E. Castro et al., Br. J. Pharmacol., 206, 833 (1992)
(36) T. Casillas et al., Biochemistry, 32, 14203 (1993)
(37) J. Pintor et al., J. Neurochem., 64, 670 (1995)
(38) E. Castro et al., J. Biol. Chem., 270, 5098 (1995)
(39) V. A. Panchenko et al., Neuroscience, 70, 353 (1996)
(40) E. Castro et al., Br. J. Pharmacol., 100, 360 (1990)
(41) J. Pintor et al., Gen. Pharmac., 26, 229 (1995)
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(43) A. Kanavarioti et al., Tett. Lett., 32, 6065 (1991)
The invention provides novel compounds of Formula I and pharmaceutical compositions thereof. The invention also provides compounds useful in the clearance of retained mucous secretion and the enhancement of ciliary beat frequency. Accordingly, a broad embodiment of the invention is directed to compounds of general Formula I or the pharmaceutically acceptable esters or salts thereof: 
wherein:
X is oxygen, methylene, difluoromethylene, imido;
n=0, 1 or 2;
m=0, 1 or 2;
n+m=0,1, 2, 3 or 4; and
B and Bxe2x80x2 are each independently a purine residue or a pyrimidine residue linked through the 9- or 1-position, respectively;
Z=OH or N3;
Zxe2x80x2=OH or N3;
Y=H or OH;
Yxe2x80x2=H or OH; provided that when Z is N3, Y is N, or when Zxe2x80x2 is N3, Yxe2x80x2 is H; and further provided that the compounds of Table I are excluded.
The compounds of the present invention are highly selective agonists of the P2Y2 and/or P2Y4 purinergic receptor; thus, they may be useful in the treatment of chronic obstructive pulmonary diseases such as chronic bronchitis, PCD, and cystic fibrosis, and may also be useful in the treatment of immobilized patients who are at risk for developing pneumonia. Furthermore, because of their general ability to clear retained mucus secretions and stimulate ciliary beat frequency, the compounds of the present invention may also be useful in the treatment of sinusitis, otitis media and nasolacrimal duct obstruction. They may also be useful for the treatment of dry eye, retinal detachment and wound healing. In addition, because of the pharmacological actions of these compounds, they are useful in facilitating sputum induction procedures. Additionally, it is postulated that the compounds of the present inventions could enhance the performance of athletes by increasing the clearance of mucous secretions from the lungs.
This invention provides novel compounds of Formula I and pharmaceutical compositions thereof. The invention also provides compounds useful in the clearance of retained mucous secretion and the enhancement of ciliary beat frequency. Accordingly, a broad embodiment of the invention is directed to novel compounds of general Formula I: 
wherein:
X is oxygen, methylene, difluoromethylene, imido;
n=0, 1 or 2;
m=0, 1 or 2;
n+m=0,1, 2, 3 or 4; and
B and Bxe2x80x2 are each independently a purine residue or a pyrimidine residue linked through the 9- or 1-position, respectively;
Z=OH or N3;
Zxe2x80x2=OH or N3;
Y=H or OH;
Yxe2x80x2=H or OH;
provided that when Z is N3, Y is H or when Zxe2x80x2 is N3, Yxe2x80x2 is H; and further provided that the compounds of Table I are excluded; or pharmaceutically acceptable esters or salts thereof.
The furanose sugar is preferably in the xcex2-configuration.
The furanose sugar is most preferably in the xcex2-D-configuration.
Preferred compounds of Formula I are the compounds of Formula IA 
wherein:
X=O;
n+m=1 or 2;
Z, Zxe2x80x2, Y and Yxe2x80x2=OH;
B and Bxe2x80x2 are uracil, thymine, cytosine, guanine, adenine, xanthine, hypoxanthine or as defined in Formulas II and III; or
X=O;
n+m=3 or 4;
Z, Zxe2x80x2, Y and Yxe2x80x2=OH;
B=uracil;
Bxe2x80x2 is uracil, thymine, cytosine, guanine, adenine, xanthine, hypoxanthine or as defined in Formulas II and I; or
X=O;
n+m=1 or 2;
Z, Y and Zxe2x80x2=OH;
Yxe2x80x2=H;
B=uracil;
Bxe2x80x2 is uracil, thymine, cytosine, guanine, adenine, xanthine, hypoxanthine or as defined in Formulas HI and III; or
X=O;
n+m=0, 1 or 2;
Z and Y=OH;
Zxe2x80x2=N3;
Yxe2x80x2=H;
B=uracil;
Bxe2x80x2=thymine; or
X=O;
n+m=0, 1 or 2;
Z and Zxe2x80x2=N3;
Y and Yxe2x80x2=H;
B and Bxe2x80x2=thymine; or
X=CH2, CF2 or NH;
n and m=1;
Z, Zxe2x80x2, Y and Yxe2x80x2=OH;
B and Bxe2x80x2 are is uracil, thymine, cytosine, guanine, adenine, xanthine, hypoxanthine or as defined in Formulas II and III;
provided that the compounds of Table I are excluded; or pharmaceutically acceptable salts thereof.
Another preferred group of the compounds of Formula I are the compounds of Formula IB or the pharmaceutically acceptable salts thereof: 
wherein:
X is oxygen, methylene, difluoromethylene or imido;
n=0 or 1;
m=0 or 1;
n+m=0, 1 or 2; and
B and Bxe2x80x2 are each independently a purine residue, as in Formula II, or a pyrimidine residue, as in Formula III, linked through the 9- or 1-position, respectively. In the instance where B and Bxe2x80x2 are uracil, attached at N-1 position to the ribosyl moiety, then the total of m+n may equal 3 or 4 when X is oxygen (see example 5). The ribosyl moieties are in the D- configuration, as shown, but may be L-, or D- and L-. The D- configuration is preferred. 
wherein:
R1 is a hydrogen, an alkyl or aryl moiety as defined below or xcfx89-A(C1-6alkyl)CONH(C1-6alkyl)-wherein A is amino, mercapto, hydroxy or carboxyl;
R2 is O (adenine 1-oxide derivatives), or is absent (adenine derivatives); or
R1 and R2 taken together form a 5-membered fused imidazole ring (1, N6-ethenoadenine derivatives), optionally substituted on the 4- or 5-positions of the etheno moiety with alkyl, aryl or aralkyl moieties as defined below;
R3 is alkyl, aryl or aralkyl, alkylamino, arylamino or aralkylamino (NHRxe2x80x2); alkoxy, aryloxy or aralkyloxy (ORxe2x80x2); alkylthio, arylthio or aralkylthio (SRxe2x80x2) as defined below; or xcfx89-A(C1-6alkyl)CONH(C1-6alkyl)B- wherein A and B are independently amino, mercapto, hydroxy or carboxyl; or pharmaceutically acceptable esters, amides or salts thereof.
Thus the substituted derivatives of adenine include adenine 1-oxide; 1,N6-(4- or 5-substituted etheno) adenine; 6-substituted adenine; or 8-substituted aminoadenine, where Rxe2x80x2 of wherein the 6- or 8-HNRxe2x80x2 groups are chosen from among: arylalkyl (C1-6) groups with the aryl moiety optionally functionalized as described below; alkyl; and alkyl groups with functional groups therein, such as: ([6-aminohexyl]carbamoylmethyl)-, and xcfx89-acylated-amino(hydroxy, thiol and carboxy)alkyl(C2-10)- and their xcfx89-acylated-amino (hydroxy, thiol and carboxy) derivatives where the acyl group is chosen from among, but not limited to, acetyl, trifluoroacetyl, benzoyl, substituted-benzoyl, etc., or the carboxylic moiety is present as its ester or amide derivative, for example, the ethyl or methyl ester or its methyl, ethyl or benzamido derivative. The xcfx89-amino(hydroxy, thiol) moiety may be alkylated with a C1-4 alkyl group.
Likewise, B or Bxe2x80x2, or both, may be a pyrimidine with the general formula of Figure III, linked through the 1- position: 
wherein:
R4 is hydrogen, hydroxy, mercapto, amino, cyano, aralkoxy, C1-6 alkylthio, C1-6 alkoxy, C1-6 alkylamino or dialkylamino, the alkyl groups optionally linked to form a heterocycle;
R5 is hydrogen, acyl (e.g., acetyl or benzoyl), C1-6 alkyl, aroyl, optionally functionalized as defined below, C1-5 alkanoyl, benzoyl, or sulphonate;
R6 is hydroxy, mercapto, alkoxy, aralkoxy, C1-6alkylthio, amino, C1-5 disubstituted amino, triazolyl, alkylamino or dialkylamino, where the alkyl groups are optionally linked to form a heterocycle or link to N3 to form an optionally substituted ring; or
R5 and R6 taken together form a 5-membered fused imidazole ring between positions 3 and 4 of the pyrimidine ring (3,N4-ethenocytosine derivatives) optionally substituted on the 4- or 5- positions of the etheno moiety with alkyl, aryl or aralkyl moieties as defined below.
R7 is hydrogen, hydroxy, cyano, nitro, alkenyl with the alkenyl moiety optionally linked through oxygen to form a ring optionally substituted on the carbon adjacent to the oxygen with alkyl or aryl groups, substituted alkynyl, halogen, substituted alkyl, perhalomethyl (e.g., CF3), C2-6 alkyl, C2-3 alkenyl, or substituted ethenyl (e.g., allylamino, bromovinyl and ethyl propenoate, or propenoic acid), C2-3 alkynyl or substituted alkynyl; or together R6-R7 may form a 5 or 6-membered saturated or unsaturated ring bonded through N or O at R6, such a ring may contain substituents that themselves contain functionalities; provided that when R8 is amino or substituted amino, R7 is hydrogen; and
R8 is hydrogen, amino or substituted amino, alkoxy, arylalkoxy, alkylthio, arylalkylthio, carboxamidomethyl, carboxymethyl, methoxy, methylthio, phenoxy or phenylthio; or pharmaceutically acceptable esters, amides or salts thereof.
In the general structures of Formula II and III above, the acyl groups advantageously comprise alkanoyl or aroyl groups. The alkyl groups which may be straight or branched advantageously contain 1 to 8 carbon atoms, particularly 1 to 4 carbon atoms optionally substituted by one or more appropriate substituents, as described below. The aryl groups including the aryl moieties of such groups as aryloxy are preferably phenyl groups optionally substituted by one or more appropriate substituents, as described below. The above-mentioned alkenyl and alkynyl groups advantageously contain 2 to 8 carbon atoms, particulary 2 to 6 carbon atoms, e.g., ethenyl or ethynyl, optionally substituted by one or more appropriate substituents as described below.
Appropriate substituents on the above-mentioned alkyl, alkenyl, alkynyl, and aryl groups are advantageously selected from halogen, hydroxy, C1-4 alkoxy, C1-4 alkyl, C6-10 aryl, C7-12 arylalkyl, C7-12 arylalkoxy, carboxy, cyano, nitro, sulfonamido, sulfonate, phosphate, sulfonic acid, amino and substituted amino wherein the amino is singly or doubly substituted by a C1-4 alkyl, and when doubly substituted, the alkyl groups optionally being linked to form a heterocycle.
The compounds of the present invention encompass their pharmaceutically acceptable esters, such as, but not limited to, acetyl and benzoyl esters. The esters may be made by reaction of the desired hydroxy compound with the appropriate acid, activated with carbonyldiimidazole, dicyclohexylcarbodiimide or other suitable condensing agent, or with an acid anhydride or acid chloride with or without a basic catalyst such as a tertary amine, quaternary amonium salt or an inorganic base.
The compounds of the present invention also encompass their non-toxic pharmaceutically acceptable salts, such as, but not limited to, an alkali metal salt such as sodium or potassium; an alkaline earth metal salt such as manganese, magnesium or calcium; or an ammonium or tetraalkyl ammonium salt, i.e., NX4+ (wherein X is C1-4). Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. The present invention also encompasses the acylated prodrugs (e.g., esters) of the compounds disclosed herein. Those skilled in the art will recognize various synthetic methodologies which may be employed to prepare non-toxic pharmaceutically acceptable salts and acylated prodrugs of the compounds encompassed by Formulas I, IA and IB.
The compounds of the present invention are highly selective agonists of the P2Y2 and/or P2Y4 purinergic receptor; thus, they are useful in the treatment of mammals including humans suffering from chronic obstructive pulmonary diseases such as chronic bronchitis, PCD, cystic fibrosis, as well as prevention of pneumonia due to immobility. Furthermore, because of their general ability to clear retained mucus secretions and stimulate ciliary beat frequency, the compounds of the present invention are also useful in the treatment of sinusitis, otitis media and nasolacrimal duct obstruction in mammals, including humans. Additionally, the compounds of the present invention are useful for treating mammals including humans with dry eye and retinal detachment.
Though the compounds of the present invention are primarily concerned with the treatment of human subjects, they may also be employed for the treatment of other mammalian subjects such as dogs and cats for veterinary purposes.
The pharmaceutical utility of compounds of this invention are indicated by the inositol phosphate assay for P2Y2 and other P2Y receptor activity. This widely used assay, as described in E. Lazarowski, et al., Brit. J. Pharm. 116, 1619-27 (1995), relies on the measurement of inositol phosphate formation as a measurement of activity of compounds activating receptors linked via G-proteins to phospholipase C.
The compounds of general Formulas I, IA, or IB may be administered orally, topically, parenterally, by inhalation or spray, intra-operatively, rectally, or vaginally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term topically as used herein includes patches, gels, creams, ointments, or nose, ear or eye drops. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In addition, there is provided a pharmaceutical formulation comprising a compound of general Formulas I, IA or IB and a pharmaceutically acceptable carrier. One or more compounds of general Formulas I, IA or IB may be present in association with one or more non-toxic pharmaceutically acceptable carriers or diluents or adjuvants and, if desired, other active ingredients. One such carrier would be sugars, where the compounds may be intimately incorporated in the matrix through glassification or simply admixed with the carrier (e.g., lactose, sucrose, trehalose, mannitol) or other acceptable excipients for lung or airway delivery.
One or more compounds of general Formulas I, IA or IB may be administered separately or together, or separately or together with mucolytics such as DNAse or acetylcysteine.
The pharmaceutical compositions containing compounds of general Formulas I, IA or IB may be in a form suitable for oral use, for example, as tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate may be employed.
Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example: sodium carboxymethylcellulose, methylcellulose and sodium alginate. Dispersing or wetting agents may be a naturally-occurring phosphatide or condensation products of an allylene oxide with fatty acids, or condensation products of ethylene oxide with long chain aliphatic alcohols, or condensation products of ethylene oxide with partial esters from fatty acids and a hexitol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anydrides. Those skilled in the art will recognize the many specific excipients and wetting agents encompassed by the general description above. The aqueous suspensions may also contain one or more preservatives, for example, ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example, sweetening, flavoring, and coloring agents, may also be present.
Compounds of general Formulas I, IA or IB may be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent. Among the acceptable vehicles and solvents that may be employed are sterile water, saline solution, or Ringer""s solution.
The compounds of general Formulas I, IA or IB may also be administered in the form of suppositories for ear, rectal or vaginal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the body temperature and will therefore melt to release the drug. Such materials are cocoa butter and polyethylene glycols.
Solutions of compounds of general Formulas I, IA or IB may be administered by intra-operative installation.
Dosage levels of the order of from about 10xe2x88x927 M to about 10xe2x88x921 M, preferably in the range 10xe2x88x925 to 10xe2x88x921M, are useful in the treatment of the above-indicated conditions. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
Compounds encompassed by the present invention may be prepared by condensation of a nucleoside mono-, di-, or triphosphate, activated with a condensing agent such as, but not limited to, carbonyldiimidazole or dicyclohexylcarbodiimide, with a second molecule of the same or a different mono-, di-, or triphosphate to form the desired dinucleotide polyphosphate; or a nucleoside phosphate, activated as above, may be condensed sequentially with a non-nucleoside mono-, di- or polyphosphate moiety, such as, but not limited to a monophosphate or pyrophosphate anion to yield the desired dinucleotide polyphosphate, the non-isolated intermediate in such a case being a mononucleotide polyphosphate; or a mono-, di- or polyphosphate moiety, activated as mentioned above, or in the form of an acid halide or other derivative reactive toward nucleophilic displacement, may be condensed sequentially with a nucleoside phosphate or polyphosphate to yield the desired dinucleotide polyphosphate; or the desired dinucleotide polyphosphate may be formed by modification a pre-formed dinucleotide polyphosphate by substitution or derivatization of a moiety or moieties on the purine, pyrimidine or carbohydrate ring. Nucleoside phosphates used as starting materials may be commercially available, or may be made from the corresponding nucleosides by methods well known to those skilled in the art. Likewise, where nucleosides are not commercially available, they may be made by modification of other readily available nucleosides, or by synthesis from heterocyclic and carbohydrate precursors by methods well known to those skilled in the art.
Those having skill in the art will recognize that the starting materials may be varied and additional steps employed to produce compounds encompassed by the present invention, as demonstrated by the following examples. In some cases protection of certain reactive functionalities may be necessary to achieve some of the above transformations. In general the need for such protecting groups will be apparent to those skilled in the art of organic synthesis as well as the conditions necessary to attach and remove such groups.